ARTICLE Received 12 Apr 2014 | Accepted 29 Sep 2014 | Published 14 Nov 2014 DOI: 10.1038/ncomms6423 Structural basis for LAR-RPTP/Slitrk complex-mediated synaptic adhesion Ji Won Um1,*, Kee Hun Kim2,*, Beom Seok Park3,*, Yeonsoo Choi4,5, Doyoun Kim5, Cha Yeon Kim6, Soo Jin Kim2, Minhye Kim1, Ji Seung Ko1, Seong-Gyu Lee4, Gayoung Choii1, Jungyong Nam4,5, Won Do Heo4,7, Eunjoon Kim4,5, Jie-Oh Lee8, Jaewon Ko1,9 & Ho Min Kim2 Synaptic adhesion molecules orchestrate synaptogenesis. The presynaptic leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) regulate synapse development by interacting with postsynaptic Slit- and Trk-like family proteins (Slitrks), which harbour two extracellular leucine-rich repeats (LRR1 and LRR2). Here we identify the minimal regions of the LAR-RPTPs and Slitrks, LAR-RPTPs Ig1–3 and Slitrks LRR1, for their interaction and synaptogenic function. Subsequent crystallographic and structure- guided functional analyses reveal that the splicing inserts in LAR-RPTPs are key molecular determinants for Slitrk binding and synapse formation. Moreover, structural comparison of the two Slitrk1 LRRs reveal that unique properties on the concave surface of Slitrk1 LRR1 render its specific binding to LAR-RPTPs. Finally, we demonstrate that lateral interactions between adjacent trans-synaptic LAR-RPTPs/Slitrks complexes observed in crystal lattices are critical for Slitrk1-induced lateral assembly and synaptogenic activity. Thus, we propose a model in which Slitrks mediate synaptogenic functions through direct binding to LAR-RPTPs and the subsequent lateral assembly of LAR-RPTPs/Slitrks complexes. 1 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea. 2 Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea. 3 Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 461-713, Korea. 4 Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea. 5 Center for Synaptic Brain Dysfunctions, Institute for Basic Science (IBS), Daejeon 305-701, Korea. 6 Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea. 7 Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 305-701, Korea. 8 Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea. 9 Department of Psychiatry, Yonsei University College of Medicine, Seoul 120-751, Korea. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.K. (email: [email protected]) or to H.M.K. (email: [email protected]). NATURE COMMUNICATIONS | 5:5423 | DOI: 10.1038/ncomms6423 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6423 euronal communication occurs at specialized asymme- resulting complex (PTPd Ig1–3/Slitrk1 LRR1). We then per- trical junctions termed synapses. Synaptogenesis, which is formed an extensive functional analysis based on this complex Nthe formation of a fully functional synapse, proceeds structure and gained important insights into the molecular via a series of neuronal process that include the initial axon– mechanism and the physiological significance of interactions dendrite contact, synapse formation and synapse maturation. between LAR-RPTPs and Slitrks in synaptogenesis. Our struc- Synaptic adhesion molecules located in the pre- and postsynaptic tural analysis of Slitrk1 LRR2 provided novel insights into how membranes play key roles in synaptogenesis. Similar to other LAR-RPTPs specifically bind to Slitrk1 LRR1 but not Slitrk1 cellular adhesion molecules (for example, those in tight junctions LRR2. Finally, from the crystal-packing lattices, we identified and immunological synapses), synaptic adhesion molecules adjacent interactions between the convex surface of Slitrk LRR1 physically align two neuronal cells and orchestrate the necessary and the neighbouring PTPd Ig1–3, and found that these lateral bidirectional neuronal signalling pathways by forming trans- interactions are essential for Slitrk1-mediated surface cluster synaptic adhesion complexes1. formation and presynaptic differentiation. These data suggest a The leukocyte common antigen-related receptor protein model of the trans-adhesion complex wherein LAR-RPTPs and tyrosine phosphatases (LAR-RPTPs), which are classified as type Slitrks coordinate synaptogenesis in a two-step process: direct IIa RPTPs, have recently emerged as a major family of synaptic binding of postsynaptic Slitrks to presynaptic LAR-RPTPs and adhesion molecules that also play crucial roles in neurite subsequent lateral assembly in the formed complexes. outgrowth, axon guidance and central nervous system (CNS) regeneration2,3. The vertebrate LAR-RPTP family has three members, LAR, PTPd and PTPs; their extracellular regions Results contain three immunoglobulin-like (Ig) domains and four to Minimal domain identification for binding and synaptogenesis. eight fibronectin III (FNIII) repeats (determined by alternative Our group and others recently demonstrated that LAR-RPTPs splicing). In particular, multiple LAR-RPTP isoforms are interact with Slitrks, and that this interaction contributes to reg- generated by alternative splicing of four mini exons, termed ulating synapse formation and function10,11.Tofurther MeA to MeD, that encode short peptides (Fig. 1a)4,5. The LAR- characterize this interaction at the molecular level, we focused on RPTPs have a single transmembrane domain followed by two PTPs and Slitrk1, a trans-synaptic adhesion molecule pair that was intracellular phosphatase domains (a membrane-proximal shown to specifically promote excitatory synapse development10. catalytic domain called D1 and a membrane-distal non-catalytic We first generated three pDis-PTPs constructs containing domain called D2) where multiple intracellular adaptor proteins different extracellular domains: the full extracellular region bind6. Recently, a growing number of postsynaptic adhesion (PTPs Full), the three Ig domains alone (PTPs Ig1–3) and the molecules have been identified as adhesion partners of the eight FNIII repeats alone (PTPs FN1–8) (Supplementary Table 1; LAR-RPTPs; these include NGL-3 (netrin-G ligand 3), TrkC see the architectures of LAR-RPTPs and Slitrks in Fig. 1a). We then (neurotrophin receptor tyrosine kinase C), IL-1RAcP (interleukin performed cell-adhesion assays with L cells expressing various 1 receptor accessory protein), IL1RAPL1 (IL-1 receptor accessory PTPs variants and the full extracellular region of Slitrk1 (Slitrk1 protein-like 1) and the Slitrks (Slit- and Trk-like family Full) (Fig. 1c,d). Consistent with our previous observation that proteins)2,3. Two alternative splicing sites within the Ig domain LAR-RPTPs mediate the trans-interaction with Slitrks10, L cells of the LAR-RPTPs (MeA and MeB) have received particular expressing PTPs full strongly aggregated with L cells expressing attention, because they modulate the interactions with various Slitrk1. The extent of aggregation among cells expressing PTPs postsynaptic ligands, such as TrkC, IL-1RAcP and IL1RAPL1 Ig1–3 was similar to that observed with PTPs Full, indicating that (refs 7–9). PTPs Ig1–3 is sufficient for Slitrk1 binding (Fig. 1c,d). PTPd Ig1–3 Among the LAR-RPTP ligands, the Slitrks were recently shown also bound to Slitrk1 (Supplementary Fig. 1). NGL-3 bound to the to induce presynaptic differentiation10,11. The six members of the FNIII repeats but not to Ig1–3, which is consistent with a previous Slitrk family (Slitrk1–6) are neuron-specific transmembrane report that NGL-3 binds to the first two FNIII repeats of LAR- proteins that localize at the postsynaptic membrane and exhibit RPTPs14. domain structures similar to those of the classic axon guidance The Slitrks contain two clusters of LRR modules, termed LRR1 molecule, Slit12. All six Slitrks possess two leucine-rich repeat and LRR2 (ref. 12) (Fig. 1a). We thus generated two additional (LRR) domains in their extracellular regions; called LRR1 and pDis-Slitrk1 constructs containing LRR1 alone (Slitrk1 LRR1) or LRR2, these domains are connected by a 70–90 amino acid loop. LRR2 alone (Slitrk1 LRR2), and performed cell-adhesion assays Each LRR domain has six LRR modules flanked by cysteine-rich (Fig. 1e,f). L cells expressing Slitrk1 Full or Slitrk1 LRR1, but not regions at its amino terminus (LRRNT) and carboxy terminus Slitrk1 LRR2, strongly aggregated with L cells expressing PTPs (LRRCT)12. The Slitrk family members exhibit synaptogenic Ig1–3, indicating that Slitrk1 LRR1 mediates PTPs binding activities through their interactions with specific LAR-RPTP (Fig. 1e,f). Consistent with the results from the cell-adhesion members10. Specifically, Slitrk1, Slitrk2, Slitrk4 and Slitrk5 assays, recombinant PTPs Ig1–3 or PTPd Ig1–3 proteins promote excitatory synapse formation by interacting with PTPs, specifically interacted with recombinant Slitrk1 Full or Slitrk1 whereas Slitrk3 enhances inhibitory synapse formation by LRR1, but not with Slitrk1 LRR2 (Fig. 1b and Supplementary interacting with PTPd10. However, the lack of a high-resolution Fig. 1c,d). In addition, the respective LRR1 domains of Slitrk2 and structure of the LAR-RPTP/Slitrk complex